I\'m developing a cell-based assay in 96-well plates that requires adherent cell

Question

I'm developing a cell-based assay in 96-well plates that requires adherent cells, as they need to be washed at least twice during the protocol. I'm using in-house strains of HT1080 cells (some overexpressing a certain protein of interest) that unfortunately have been selected for suspension growth in chemically-defined media (50/50 CD-CHO/CD-293). I can convince them to adhere by culturing in DMEM with 10% FBS and growing them in CELLBind flasks, but they still tend to pull away if there is very much shearing force, such as during the wash steps, or when they're in PBS during the assay wash and incubation steps (maybe 2 hours total for now, I'm still optimizing that).

Unfortunately, the parameters of my experiments don't allow me to keep the cells in serum-containing media during the assay, as it would interfere with the uptake of my drug. Like I've said, when in PBS they start detaching, and I would assume the same in HBSS, which I don't really want to use anyway due to its low buffering capacity in CO2 incubators.

Might DMEM alone (without FBS) provide a little more impetus to stay attached during the assay? What would the effect be of increasing the amount of FBS to say 15 or 20% during culturing - would that promote stronger attachment? I'm a little hesitant to coat the wells with poly-D-lysine, as my readout is on a fluorescent imager, and I'm worried it would substantially increase background. I don't really have enough time to re-select the cells for adherent growth (although if I'd known about these issues from my predecessor 3 or 4 months ago I would have started right away...), so are there any other tricks I can try in the meantime? Are there any media supplements I could use to promote adherin expression or something like that? Any tips or advice at all would be appreciated.

Explanation / Answer

My main concern here is why you are using suspension-culture selected cells for an adherent cell-based assay? Is it possible to obtain a clone of adherent HT1080? Depending on what you are assaying with your protein of interest, the cell biology may be altered changing from suspension/adherent culture systems.

Here are my experiences using PC12 cells in culture, which are weakly adherent cells, with a preference to grow in clumps, rather than monolayers. I have found that these cells have a preference for specific tissue plastic; (without mentioning names), these cells will adhere on some poly-D-lysine coated plates while they won't adhere on others. I routinely perform immunofluorescence microscopy with these cells, and grow them on glass cover slips which I have skim-coated with rat tail type I collagen. This coating provides minor background in the green range and only when imaging close to the glass. With a bright fluorophore (e.g., AlexaFluor or cyanin dyes for fixed cell; enhanced GFP for live cells), this backgorund is not a problem for quantitative microscopy.

Lastly, for secretion assays we use high-glucose DMEM supplemented with 1% dialyzed FBS. If memory serves, this retains all serum components less than ~15 kDa, which includes various growth factors. I have observed these cells continue to live for up to one day (and maybe longer?) in supplemented medium, while they die after a few hours without. This may also help with non-specific binding you may be worried about with drug addition.

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